Malaria caused by the protozoan parasite, Plasmodium falciparum is estimated to cause 1-2 million deaths a year. One of the major barriers to its control is the parasite's complex life cycle; each developmental stage is distinct and can respond differently to interventions. Therefore, it is important to have a comprehensive understanding of the molecular events involved throughout the life cycle. Transmission from one person to another requires that mature intraerythrocytic sexual-stage parasites, gametocytes, be taken up from an infected human host by a mosquito during a blood meal. This transition stimulates the gametocytes to emerge from erythrocytes and over the next 24 hours to develop into oocysts that produce thousands of sporozoites that are infectious to humans. We have demonstrated that the stimulation of emergence results in the proteolytic processing of gametocyte surface protein Pfs230 by a cysteine protease and our collaborators have preliminary evidence that the cysteine protease inhibitor, E64d blocks the ability of the parasite to infect mosquitoes. Recently, we have found that the mRNA encoding a cysteine protease, falcipain 1 is up regulated in gametocytes. Transcripts for two additional falcipain cysteine proteases, falcipain 2 and 3, are also present in gametocytes, as well as asexual parasites. Three additional genes have been identified in the P. falciparum genome that have homology to cysteine proteases, PfB0325c, PfB0330c, PfB0335c. We hypothesize that at least one cysteine protease is required for transmission of P. falciparum to the mosquito and propose to identify and localize the cysteine proteases expressed by gametocytes (Specific Aim 1). Antibodies specific for these proteases will be used to determine their subcellular localization throughout emergence and this data compared with the emergence-induced changes in proteins associated with the gametocyte parasitophorous vacuole membrane or the plasma membrane of the gametocyte or erythrocyte. The function and targets of cysteine proteases with expression patterns consistent with a role in gametogenesis, fertilization or subsequent development in the mosquito will be analyzed further by targeted gene disruption and inhibitor studies (Specific Aims 2 & 3). In addition to increasing our understanding of the genes involved, this information can be used to direct the design of cysteine protease inhibitors that will effectively block malaria transmission.